The present invention relates to a biosensor that ensures rapid and highly accurate quantification of a specific component contained in a sample by a simplified procedure, and more specifically to a method of forming a reaction layer of the biosensor.
One proposed biosensor adopts the simple technique that quantifies a specific component in a sample without diluting or stirring a sample solution (Japanese Laid-Open Patent Publication No. Hei 2-062952).
This prior art biosensor is manufactured by forming an electrode system, which includes a measuring electrode, a counter electrode, and a reference electrode, on an electrically insulating base plate by a known method like screen printing and further forming an enzyme reaction layer, which includes a hydrophilic polymer, an oxidoreductase, and an electron mediator, on the electrode system. A buffer may be added to the enzyme reaction layer according to the requirements.
When a sample solution including a substrate is dropped on the enzyme reaction layer of the biosensor manufactured in the above manner, the enzyme reaction layer is dissolved to make the enzyme react with the substrate and reduce the electron mediator. After completion of the enzyme reaction, the concentration of the substrate included in the sample solution is determined, based on the observed oxidation current flowing in the process of electrochemically oxidizing the reduced electron mediator.
The following describes a glucose sensor as one example of the biosensor.
A generally known method of quantifying glucose combines glucose oxidase with either an oxygen electrode or a hydrogen peroxide electrode.
Glucose oxidase selectively oxidizes a substrate xcex2-D-glucose to D-glucono-xcex4-lactone with oxygen as the electron mediator. In the course of this reaction, oxygen is reduced to hydrogen peroxide. Glucose is quantified by measuring the quantity of oxygen consumption due to the reduction with the oxygen electrode or by measuring the quantity of hydrogen peroxide production with the hydrogen peroxide electrode, such as a platinum electrode.
The quantification of some substrates of interest according to this prior art method is, however, significantly affected by the concentration of dissolved oxygen. The measurement is unavailable in the absence of oxygen. Another type of the glucose sensor has accordingly be developed, which does not use oxygen as the electron mediator but utilizes a metal complex or an organic compound, such as potassium ferricyanide, a ferrocene derivative, or a quinone derivative, for the electron mediator.
The glucose sensor of this type oxidizes the reduced form electron mediator, which results from the enzyme reaction, on an electrode and determines the concentration of glucose from the observed oxidation current.
The biosensor according to this technique is, in principle, applicable to measurement of various substances by using an enzyme that acts upon each substance of interest as the substrate.
For example, application of cholesterol oxidase or cholesterol dehydrogenase and cholesterol esterase for the oxidoreductase enables measurement of serum cholesterol, which is used as a diagnostic indication in a diversity of medical institutes.
The progress of the enzyme reaction of cholesterol esterase is remarkably slow. Addition of an appropriate surface active agent enhances the activity of cholesterol esterase and shortens the time required for the whole reaction.
In this prior art biosensor, for example, a reaction layer is obtained by dissolving potassium ferricyanide, which is one of the electron mediators discussed above, alone or with other components in a solvent, dropping the solution in a desired area for the reaction layer on a base plate, and drying the dropped solution with warm blast. In this reaction layer, potassium ferricyanide deposits in the form of needles having the longitudinal dimension of even greater than 1 mm. The reaction layer accordingly has the heterogeneous configuration, which worsens the measurement accuracy of a resultant sensor.
Compared with that in the glucose sensor, the reaction layer in the cholesterol sensor contains a higher concentration of the corresponding enzyme. The prior art method that forms such a reaction layer by drying the dropped solution with warm blast causes the resultant reaction layer to be slowly dissolved in a sample solution and have poor response.
The object of the present invention is thus to provide a biosensor that has high accuracy and excellent response to a substrate even in a high concentration range of the substrate.
In order to form a reaction layer containing a reagent such as potassium ferricyanide, which tends to deposit from an aqueous solution in the form of crystals, or a high concentration of an enzyme, the inventors have found the suitable method that dissolves a material constituting a target reaction layer in a solvent of a sublimable substance to prepare a solution, applies the solution in a desired area to form the target reaction layer, freezes the applied solution, and sublimates the solvent included in a solid matter of the frozen solution under reduced pressure for removal. This method gives the reaction layer that has a large surface area and is easily dissolved i n a sample solution. The technique of the present invention is based on these findings.
At least part of the a above and the other related objects of the present invention is attained by a method of manufacturing a biosensor, which includes an electrically insulating base plate, an electrode system that is provided on the electric ally insulating base plate and includes a measuring electrode and a counter electrode, and a reaction reagent system that includes at least an oxidoreductase and an electron mediator as reagents. The reagents of the reaction reagent system are present as a reaction layer structure that includes at least one reaction layer and is formed on or in the vicinity of the electrode system . The method includes the step of forming a specific reaction layer of the reaction layer structure that contains at least one specific reagent of the reaction reagent system. The step comprises:
(1) dissolving the at least one specific re agent in a solvent of a sublimable substance to prepare a solution;
(2) applying the solution in a desired area to form the specific reaction layer;
(3) freezing the applied solution; and
(4) sublimating the solvent included in a solid matter of the frozen solution under reduced pressure for removal.
In accordance with one preferable application of the present invention, the specific reaction layer contains all the reagents of the reaction reagent system.
In accordance with another preferable application of the present invention, the reaction layer structure has a plurality of reaction layers and only the specific reaction layer contains the at least one specific reagent of the reaction reagent system.
In accordance with still another preferable application of the present invention, the method includes the step of forming a stack of plural reaction layers as the reaction layer structure, wherein the at least one specific reagent of the reaction reagent system is contained only in an upper-most reaction layer of the stack. The step includes the sub-steps of: pre-forming the stack of plural reaction layers except the upper-most reaction layer; dissolving the at least one specific reagent in a solvent of a sublimable substance to prepare a solution and applying the solution on the pre-formed stack of plural reaction layers without the upper-most reaction layer; freezing the applied solution; and sublimating the solvent included in a solid matter of the frozen solution under reduced pressure for removal.
It is preferable that the method further includes the steps of: forming a cover member on the electrically insulating base plate, which is joined with the base plate to define a sample solution supply pathway, through which a sample solution flows to the electrode system; causing the reaction layer structure to be exposed to the sample solution supply pathway; and forming at least one reaction layer of the reaction layer structure on the cover member.
In the case where the oxidoreductase is an enzyme functioning as a catalyst of the oxidation reaction of cholesterol, it is preferable that the electron mediator is included in another reaction layer different from a reaction layer containing the enzyme.
In accordance with another preferable application of the present invention, the oxidoreductase is an enzyme functioning as a catalyst of the oxidation reaction of cholesterol, and the electron mediator is contained in another reaction layer different from a reaction layer containing the enzyme.
In accordance with still another preferable application of the present invention, the oxidoreductase is an enzyme functioning as a catalyst of the oxidation reaction of cholesterol, and the layer containing the enzyme further contains a surface active agent.
In accordance with one preferable application of the present invention, the above-mentioned at least one specific reagent of the reaction reagent system is an oxidoreductase or potassium ferricyanide which is an electron mediator.
It is also preferable that at least lower-most reaction layer of the reaction layer structure includes a hydrophilic polymer.
While the novel features of the invention are set forth particularly in the appended claims, the invention, both as to organization and content, will be better understood and appreciated, along with other objects and features thereof, from the following detailed description taken in conjunction with the drawings.
FIG. 1 is a disassembled perspective view illustrating the structure of a biosensor without a reaction layer structure in one embodiment of the present invention.
FIG. 2 is a vertical sectional view illustrating a main part of the biosensor shown in FIG. 1.
FIG. 3 is a vertical sectional view illustrating a main part of another biosensor in another embodiment of the present invention.
FIG. 4 is a graph showing responses of cholesterol sensors prepared as examples of the present invention and comparative examples.